Revealing the unknown aspects of initial steps of prenylated flavin mononucleotide biosynthesis – the role of Lys129 in PaUbiX

Background Prenylated flavin mononucleotide (prFMN) is a recently discovered, heavily modified flavin compound. It is the only known cofactor that enables enzymatic 1,3-dipolar cycloaddition reactions. It is produced by enzymes from UbiX family, from flavin mononucleotide and either dimethylallyl mono- or diphosphate. prFMN biosynthesis is currently reported to be initiated by a protonation of the substrate by Glu140. Methods Computational chemistry methods are applied herein - mostly different flavors of molecular dynamics MD, such as Constant pH MD, hybrid Quantum-Mechanical / Molecular Mechanical MD, and classical MD. Results Glu140 competes for a single proton with Lys129 but it is the latter that adopted a protonated state throughout most of the simulation time. Lys129 plays a key role in the positioning of the DMAP’s phosphate group within the PaUbiX active site. DMAP’s breakdown into a phosphate and a prenyl group can be decoupled from the protona-tion of the DMAP’s phosphate group. Conclusions The role of Lys129 in functioning of PaUbiX is reported for the first time. The severity of interactions between Glu140, Lys129, and DMAP’s phosphate group enables an unusual decoupling of phosphate’s protonation from DMAP’s breakdown. Those findings are most likely conserved throughout the UbiX family to the structural re-semblence of active sites of those proteins. Significance Mechanistic insights into a crucial biochemical process, biosynthesis of prFMN, are provided. This study, alt-hough purely computational, extends and perfectly complements the knowledge obtained in classical laboratory experiments.